Antibodies against CD3 are a central element in many T cell proliferation protocols. Immobilized on a surface, anti-CD3 delivers an activating and proliferation-inducing signal by crosslinking of the T cell receptor complex on the surface of T cells. By immobilizing anti-CD3 and anti-CD28 to simultaneously deliver a signal and a co-stimulatory signal, proliferation can be increased (Baroja et al (1989), Cellular Immunology, 120: 205-217). In WO09429436A1 solid phase surfaces such as culture dishes and beads are used to immobilize the anti-CD3 and anti-CD28 antibodies. Regularly, the immobilization on beads is performed on DynaBeads® M-450 having a size of 4.5 μm in diameter.
EP01257632B1 describes a method for stimulating a population of T-cells by simultaneous T-cell concentration and cell surface moiety ligation that comprises providing a population of cells wherein at least a portion thereof comprises T-cells, contacting the population of cells with a surface, wherein the surface has attached thereto one or more agents that ligate a cell surface moiety of at least a portion of the T-cell and stimulates at least that portion of T cells or a subpopulation thereof and applying a force that predominantly drives T-cell concentration and T-cell surface moiety ligation, thereby inducing T-cell stimulation. The term force as used herein refers to a force used to drive the cells and may include a variety of forces that function similarly, and include a force greater than gravitational force, a hydraulic force, a filtration force generated by transmembrane pressure, a centrifugal force, or a magnetic force. EP1257632B1 describes that ratios of particles to cells can vary, however certain preferred values include at least 1:4 to 6:1, with one preferred ratio being at least 2:1 beads per T-cell. Regularly, DynaBeads® M-450 having a size of 4.5 μm in diameter coupled to anti-CD3 and anti-CD28 antibodies were used in experiments in a bead/T-cell ratio of 3:1. Again, these methods use solid phase surfaces to co-immobilize T cell stimulation agents such as anti-CD3 and anti-CD28 antibodies. These surfaces are cell-sized and comparable with the T cells themselves.
US2008/0317724A1 discloses that the spatial presentation of signal molecules can dramatically affect the response of T cells to those signal molecules. For example, when anti-CD3 and anti-CD28 antibodies are placed on separate predefined regions of a substrate, T cells incubated on the substrate secrete different amounts of interleukin-2 and/or exhibit spikes in calcium, depending not only on the types but also on the spacing of these signal molecules. For example, a pattern was generated with anti-CD3 and anti-CD28 antibodies, where anti-CD3 antibodies occupied a central feature surrounded by satellite features of anti-CD28 antibodies that were spaced about 1 to 2 microns from the central anti-CD3 feature. When the anti-CD28 antibody features were spaced about 1 to 2 microns apart, the T cell secretion of interleukin-2 (IL-2) was enhanced compared to when the anti-CD3 and anti-CD28 antibodies were presented together to the T cells in “co-localized” features.
The publication of Erin R Steenblock and Tarek M Fahmy (Molecular Therapy vol. 16 no. 4, 765-772 April 2008) uses solid-surface nanoparticles (130 nm) and show that these nanoparticle stimulate T cells weaker than microparticles (8 μm). The authors stated that these findings are supported by those of previous reports (Mescher, M F (1992). J Immunol 149: 2402-2405.), demonstrating that micron-sized particles, which are close in size to T cells, provide optimal T-cell stimulation. Mesher's study demonstrated the critical importance of a large, continuous surface contact area for effective CTL activation. Using class I alloantigen immobilized on latex microspheres, particle sizes of 4 to 5 microns were found to provide an optimum stimulus. Below 4 microns, responses decreased rapidly with decreasing particle size, and large numbers of small particles could not compensate for suboptimal size.
U.S. Pat. No. 8,012,750B2 discloses a biodegradable device for activating T-cells. The biodegradable support is first formulated into a shape, such as a microsphere. The biodegradable supports then coated with a first material providing a reactive surface which is capable of binding to second materials. The second materials have a reactive surface which permits binding to surface structures on a cell. The biodegradable support can be formulated into various shapes. Microspheres are a preferred formulation because of the simplicity of manufacture and the spherical shape allows an increased surface area for interaction with cellular receptors. According to U.S. Pat. No. 8,012,750B2 nanospheres do not provide enough cross-linking to activate naive T-cells and thus can only be used with previously activated T-cells. Again, experimental data were generated with spheres co-immobilized with anti-CD3 and anti-CD28 antibodies ranging in size from 4 to 24 microns with a mean of 7 microns.
Taken together, beads or microspheres used in the state of the art for T cell activation via immobilized T cell stimulatory antibodies are cell-sized (mostly 1 to 10 μm in size), uniformly round-shaped particles. Beads of this size have several disadvantages with regard to their potential to interact with T cells as well as their production, handling and safety in clinical T cell therapy procedures.                1. Due to the solid surface of the bead the size of interaction area between the bead and cells is limited.        2. Their preparation is complex and costly as compared to soluble antibodies and it is especially inconvenient to generate them in cGMP quality, e.g. due to their size no sterile filtration is possible, sedimentation complicates handling, i.e. constant particle number/volume during filling and antibody loading.        3. They are inconvenient to use for in vitro processes to generate T cell therapeutics for in vivo use,                    since they have to be added to cells in defined cell/bead ratios at defined density cell/beads per surface area,            adaption of stimulation strength is only possible to some extent, since the T cell stimulation strength is mostly determined by the density of antibodies on the cell surface and not by the number of beads/cell            aliquoting is inaccurate due to sedimentation,            sterile filtration is not possible            due to their size they might affect cell viability and function and they cannot simply be removed from cells by centrifugation. Therefore either special protocols for “bead removal” or biodegradable particles have been developed. However both methods suffer from inaccuracies with regard to the actual number of residual beads after the removal process, leaving behind a certain risk for toxic effects if T cell stimulatory beads are injected into patients. This problem is particularly relevant because of the size of the particles, since each single particle on its own might still have retained the capacity to activate T cells in vivo.                        
Therefore, there is a need for an improved in-vitro method for T cell stimulation.
All references cited herein, including patent applications and publications, are incorporated by reference in their entirety.